DNA damage repair pathways are critical for maintaining genome stability and preventing mutations in proto- oncogenes or tumor suppressor genes that then drive the development of cancer. Understanding DNA repair pathways is also critical given that many chemotherapeutic agents function by damaging DNA. In this way DNA repair pathways not only influence cancer susceptibility but also affect the efficacy of cancer chemotherapy. The DNA mismatch repair (MMR) pathway functions to repair base pair mismatches and small insertion/deletion mispairs that occur during normal DNA replication. Defects in MMR result in increased mutation rates and lead to cancer predisposition syndromes, such as Lynch syndrome, and sporadic tumors. Unlike the well-defined E. coli MMR pathway, the exact mechanisms of repair downstream of mispair recognition in eukaryotic MMR are still unclear. Our recent experiments in S. cerevisiae have uncovered the existence of at least two MMR sub pathways: 1) an Exonuclease1 (Exo1)-independent pathway that appears to be coupled to DNA replication and 2) an Exo1-dependent pathway. Little is known about the specific mechanisms of these MMR subpathways or their impact on human cancer development or response to chemotherapy. PCNA (Proliferating Cell Nuclear Antigen) is an integral part of the MMR pathway, although its mechanistic roles are not completely understood. PCNA is required for DNA synthesis after excision of the mispair, and plays multiple roles in upstream repair steps that potentially dictate MMR sub pathway function. This project will generate a collection of separation-of-function mutations in POL30, which encodes PCNA, that cause dysfunction in either the Exo1-independent or Exo1-dependent sub pathways of MMR by using targeted genetic screening. These mutations will be characterized with functional biochemical and cell biological assays to dissect the mechanistic roles of PCNA within each sub pathway and to illuminate the poorly understood details of eukaryotic MMR downstream of mispair recognition. This mechanistic data will be used to guide future experiments into how PCNA mutations and MMR sub pathway defects influence human tumor formation and chemotherapy resistance.